1. Field of the Invention
The present invention relates to bird gender identification, and in particular relates to Columbidae gender molecular identification.
2. Description of the Related Art
Gender identification is especially important to monitor the population size in areas that have decreased population trends. The sex ratio is an important factor in breeding birds, monitoring population growth, and detecting global changes in relationships. However, determining the sex of a bird belonging to the Columbidae family is difficult because most are monomorphic.
Traditionally, molecular gender identification of birds is based on the differences in length of intron between the chromo-helicase-DNA binding protein (CHD)-Z and CHD-W genes amplified by the Griffiths P2/P8 primer set (Griffiths R, Double M C, Orr K, Dawson R J. (1998) A DNA test to sex most birds. Molecular Ecololgy 7:1071-5), i.e., males contain a single CHD-Z band (ZZ) and females contain two bands (CHD-Z and CHD-W; ZW), after electrophoresis. Currently, other methods such as re-designed non-P2/P8 primers for polymerase chain reaction (Fridolfsson, A. and Ellegren, H. (1999) A simple and universal method for molecular sexing of non-ratite birds. Journal of Avian Biology, 30, 116-121.; Chang, H. W., Chou, T. C., Gu, D. L., Cheng, C. A., Chang, C. C., Yao, C. T., Chuang, L. Y., Wen, C. H., Chou, Y. C., Tan, K. Y. et al. (2008) An improved polymerase chain reaction method for gender identification of eagles. Molecular and cellular probes, 22, 184-188.), polymerase chain reaction-RFLP (Sacchi, P., Soglia, D., Maione, S., Meneguz, G., Campora, M. and Rasero, R. (2004) A non-invasive test for sex identification in short-toed Eagle (Circaetus gallicus). Molecular and cellular probes, 18, 193-196.; Reddy, A., Prakash, V. and Shivaji, S. (2007) A rapid, non-invasive, polymerase chain reaction-based method for identification of sex of the endangered Old World vultures (white-backed and long-billed vultures)—Implications for captive breeding programmes. Current Science 2007; 92:659-62), RAPD-polymerase chain reaction (Wu, C. P., Horng, Y. M., Wang, R. T., Yang, K. T. and Huang, M. C. (2007) A novel sex-specific DNA marker in Columbidae birds. Theriogenology, 67, 328-333.) and AFLP-polymerase chain reaction (Huang, C. W., Cheng, Y. S., Rouvier, R., Yang, K. T., Wu, C. P. and Huang, M. C. (2007) AFLP fingerprinting for paternity testing in ducks. Br Poult Sci, 48, 323-330.) have been developed for the gender identification of birds. However, all require the gel electrophoresis step after the polymerase chain reaction and are not robust for gender identification of birds in a high-throughput format.
Alternatively, melting curve analysis is capable of simultaneously measuring the melting temperature (Tm) for many polymerase chain reaction amplicons. The melting curve analysis does not require the electrophoresis step; thereby saving time and having high-throughput. Melting curve analysis is widely used in many fields such as for the detection of methylation, SNP genotyping and mutation, genus identification of microbials, quantification of chromosome conformation capture. However, melting curve analysis is seldom applied in the gender identification of birds.
For melting curve analysis, different Tm values for the specific polymerase chain reaction amplicons are analogous to the different shift mobilities for the corresponding electrophoretic bands. Amplicons with different lengths have different melting temperatures (Tm), ie. the longer the length is, the greater the Tm value is, and the shorter the length is, the lower the Tm value is. Therefore, it is possible that the gender of some Columbidae species may be identified using melting curve analysis based on the different Tm values of their CHD-Z and CHD-W amplicons. Although melting curve analysis may be used to determine Tm values for CHD-Z and CHD-W amplicons, their differences in length may vary from species to species. Accordingly, melting curve analysis for species wherein the difference in length of intron for CHD-Z and CHD-W amplicons is small, which results in a small Tm value difference therebetween, is not applicable as the degree of resolution for real-time polymerase chain reaction machines may be insufficient.
Recently, for gender identification of three species belonging to the Columbidae family, a female-specific primer generated from an RAPD method has been disclosed (Wu, C. P., Horng, Y. M., Wang, R. T., Yang, K. T. and Huang, M. C. (2007) A novel sex-specific DNA marker in Columbidae birds. Theriogenology, 67, 328-333.). The polymerase chain reaction product for the lengths of 777-, 778-, and 770-bp, for three species belonging to the Columbidae family such as Streptopelia orientals, Streptopelia chinensis and Columba livia, have been respectively generated. Also, 16S rRNA (256-bp) has been used as a polymerase chain reaction control for dove gender identification using the female-specific primer. However, if DNA is degraded or the quality of DNA from samples collected during field research is poor, it may be difficult to amplify a long polymerase chain reaction product that required by the female-specific primer. Therefore, it is possible that some degraded and shorter female DNA samples may not be correctly detected using the female-specific primer. Specifically, if there is only one copy for nucleus genes, the number of mitochondrial may be 1000. Thus, the amount of copies of mitochondrial genes may be about 1000 times that of the nucleus genes. Therefore, as long as one of the 1000 mitochondrials is complete, amplification of the 16S rRNA thereof is possible, i.e., a female may be regarded as a male as their 16S rRNA would be positive and the female-specific polymerase chain reaction would be negative.
Therefore, a high-throughput, and precisely accurate method for Columbidae gender identification is currently needed.